The broad long-term objective of this research proposal is to prevent or lessen the effects of pulmonary barotrauma. Four to fifteen percent of ventilated patients experience pneumothorax due to barotrauma, a lung injury associated with high pressures or volumes produced by mechanical ventilation. In addition to pneumothorax, an easily-recognized hallmark of barotrauma, recent evidence suggests that mechanical ventilation may induce profound changes in lung fluid balance and blood-gas barrier permeability. Experiments have shown that lung injury is actually more closely associated with high volumes rather than high pressures, suggesting that deformation of the fine structure of the lungs parenchyma makes a critical contribution to the injury. During the first period of funding, the hypothesis that the alveolar epithelial lining of the lung is vulnerable to injury from large-amplitude dynamic deformations, comparable to those experienced during mechanical ventilation was confirmed, and these findings were supported by recent clinical studies demonstrating a significant reduction in mortality with low tidal volume ventilation. These findings emphasize the importance of experimental models to determine the mechanisms that make the epithelium susceptible to mechanical deformation, and to define interventions that protect the epithelium against mechanical deformation. Using our established in vitro model for VILI, we propose to extend our understanding of deformation-induced epithelial injury at the cellular and molecular level. The overall hypothesis of the research plan is that mechanical strain is the primary cause of acute alveolar epithelial functional alterations in VILI. The short-term objectives of this 5-year plan are I) to measure the strains associated with alterations in paracellular (Aim I) and transcellular (Aim 2) epithelial transport, and 2) to determine molecular pathways responsible for transducing mechanical signals within the cell (Aim 3). Thus, the proposed research plan will lay the foundation for the development of pharmacological therapies to prevent or ameliorate VILI. The planned experimental studies are essential to address our long-term goal of developing safer, more effective mechanical ventilator modes and new intervention strategies to prevent acute VILI due to epithelial over-distention.